A fascinating question in Vision Research is how the lens, a tissue made of concentric layers of living fiber cells, maintains both transparency and homeostasis in the absence of blood vessels and for the life of the animal. In the current model, a primitive "circulatory system" comprised of channels and transporters is thought to move water, ions, and nutrients, and thereby maintain the metabolic integrity of the lens interior. A high concentration (450 mg/ml) of soluble proteins of the crystallin family forms a gel-like structure responsible for the refractive properties yielding transparency. Disruption of either the system of channels or the assemblies of crystallins results in a loss of transparency and the formation of cataracts. This application proposes to determine the 3D-structure and protein composition of the macromolecular assemblies underpinning lens transparency and cataract formation in vertebrate lenses. To accomplish this goal, experiments are proposed to determine the 3D-structure of these assemblies, using the newly perfected techniques of Conical Electron Tomography and Density Segmentation. This approach will allow us for the first time to determine the changes in the 3D- structure of assemblies at high resolution (~3 nm), in their cellular environments and without the need to impose symmetry or to average over many molecules. The three aims comprising the application will focus specifically on: a) membrane-bound assemblies comprised of AQP0, Cx46 and Cx50, b) "beaded" filaments of the fiber cytoskeleton, composed of filensin and phakinin, and c) assemblies comprised of 1-crystallin. Changes in the 3D-structure of these assemblies that are induced by protein phosphorylation, a single amino acid replacement in the central domain of the 1-crystallin (1A-Y118D) that induces a nuclear cataract in rodents and humans will be studied using Conical Tomography and Density Segmentation. We expect that a better understanding of the changes in the 3D-structure and protein composition of these assemblies will lead to strategies that might slow down or prevent the onset of cataract. PUBLIC HEALTH RELEVANCE: Cataracts are the principal cause of blindness worldwide. Once the disease develops, it can only be treated by surgery. In the last year alone, almost three million Americans required surgical procedures to remove lenses and restore vision, with expenses in the billions. The identification of agents that might slow down or completely prevent the appearance of the disease requires a detailed understanding of the 3D-structure and protein composition of the assemblies responsible for lens transparency.